The ability to detect, characterize, and manipulate biomolecules in complex media is critical for understanding biochemical and metabolic processes. Methods and systems which are capable of detecting trace amounts of microorganisms, pharmaceuticals, hormones, viruses, antibodies, nucleic acids and other proteins have been based on well known binding reactions, e.g. antigen-antibody reactions, nucleic acid hybridization techniques, and protein-ligand systems. Much attention is being given to the design, synthesis, and employment of molecular probes of enzyme structure and function [Wilker, J. J. et al. Angew. Chem. Int. Ed. (1999), 38, 90–92; Hamachi, I. et al., J. Am. Chem. Soc. (1999), 121, 5500–5506; Dmochowski, I. J., et al. Proc. Natl. Acad. Sci. USA (1999), 96, 12987–12990; Atkinson, R. N. et al. J. Org Chem. (1999), 64, 3467–3475; Tschirret-Guth, R. A. et al. J. Am. Chem. Soc. (1999), 121, 4731–4737; DiGleria, K. et al. J. Am. Chem. Soc. (1998), 120, 46–52; Murthy, Y.; Massey, V. Meth. Enzymol. (1997), 280, 436–460; Newcomb, M. et al. J. Am. Chem. Soc. (1995), 117, 3312–3313; Atkinson, J. K.; Ingold, K. U. Biochemistry (1993), 32, 9209–9214; Liu, K. E. et al., J. Am. Chem. Soc. (1993), 115, 939–947; Tschirret-Guth, R. A. et al. J. Am. Chem. Soc. (1998), 120, 7404–7410], owing in part to the abundance of naturally occurring cavity proteins [Tainer, J. A. et al. J. Mol. Biol. (1982), 160, 181–217; Bigler, T. L. et al., Prot. Sci. (1993), 2, 786–799; Badger, J. et al., Proc. Natl. Acad. Sci. USA (1988), 85, 3304–3308; Poulos, T. L. et al., Biochemistry (1986), 25, 5314–5322] and in part to the power of site-directed mutagenesis, to modify existing cavities and create new substrate binding sites [Wilcox, S. K. et al., Biochemistry (1998), 37, 16853–16862; Goldsmith, J. O. et al., Biochemistry (1996), 35, 2421–2428; DePillis, G. D. et al., J. Am. Chem. Soc. (1994), 116 6981–6982; Fitzgerald, M. M. et al., Biochemistry (1994), 33, 3807–3818; Eriksson, A. E. et al., Nature (1992), 355, 371–373].
Typically, detection of biomolecules of interest is performed by an observable tag or label attached to one or more of the binding elements (i.e. substrates) of the biomolecule and indicated by the presence or absence of the observable tag. Of particular interest are labels that can emit energy as luminescence through photochemical, chemical, and electrochemical processes.
The detection of specific proteins through luminescence spectroscopy should be useful in a wide variety of fields. The rise of combinatorial chemistry has necessitated the development of sensitive and rapid screens for drug-target interactions. Luminescence is ideal for rapid screening because of its speed and sensitivity. Similarly, a luminescent probe for the in vivo detection of enzyme expression and localization is generally useful. Examples of widely used probes include small molecule detectors for mono- and divalent cations and Green Fluorescent Protein hybrid proteins. (d. Silva, A. P. et al., Coord. Chem. Rev. (1999) 185–186, 297–306; Tsien, R. Y. Annu. Rev. Biochem. (1998) 67, 509–544; Takahashi, A. et al., Physiol. Rev. (1999) 79, 1089–1125). The wide usage of these techniques suggests that a method of detecting the localization and concentration of a given enzyme is highly desirable. However, few techniques currently exist that take advantage of the inherent specificity of an enzyme for its substrate.
In addition, molecules with photosensitizers attached to cofactors [Hamachi, I. et al., J. Am. Chem. Soc. (1999), 121, 5500–5506] can rapidly deliver redox equivalents to buried active sites for potential therapeutic applications.
Particularly important target biomolecules are oxygenases (e.g. cytochrome P450) involved in drug metabolism and many disease states, including liver and kidney dysfunction, neurological disorders, and cancer. 54 human cytochrome P450 genes have been identified. The cytochrome P450 genes are broken down into many families and subfamilies. The first isolated human P450s were 1A1, 1A2, 2A6, 2C8, 2C9, 2D6, 2E1, 3A4, 3A5, and 4A11. The 1A family, for example, is actively studied due to its role in carcinogen activation (F. P. Guengrich, “Human Cytochrome P450 Enzymes” in Cytochrome P450: Structure, Mechanism, and Biochemistry, 2nd ed. Ed. Paul R. Ortiz de Montellano, Plenum Press, New York, 1995, pp. 473–536.) and would be an optimal target for characterization.
Although more than 100 mammalian microsomal P450 isozymes have been identified, direct information about their structures and physiological function is lacking. The best characterized of these is, cytochrome P450cam(P450). Crystal structures are available for only six P450 oxygenases (Poulos, T. L., et al. (1995) in Cytochrome P450: Structure, Mechanism, and Biochemistry, 2nd edn, ed. Ortiz de Montellano, P. R. (Plenum Press, New York), pp. 125–150), all but one of which are water-soluble bacterial enzymes.
New methods for detecting mammalian P450s and characterizing their structures (Tschirret-Guth, R. A., et al. (1999) J. Am. Chem. Soc. 121, 4731–4737) would facilitate rational drug design (Ortiz de Montellano, P. R. & Correia, M. A. (1995) in Cytochrome P450: Structure, Mechanism, and Biochemistry, 2nd edn, ed. Ortiz de Montellano, P. R. (Plenum Press, New York), pp. 305–364) and the engineering of new catalysts (Joo, H., Zhanglin, L. & Arnold, F. H. (1999) Nature 399, 670–673; Stevenson, J.-A., et al. (1996) J. Am. Chem. Soc. 118, 12846–12847) for use in diagnosis and/or therapy of diseases.
Another luminescent ruthenium complex [Ru(phen)2dppz]2+ is nearly undetectable in water but moderate in non-aqueous solvents. (Chambon, J.-C. et al., New J. Chem. (1985) 9, 527–529) The discovery that this and similar compounds also emit light when intercalated into doubled stranded DNA resulted in publications, both on the original dppz complexes and on related compounds. (Friedman, A. E. et al., J. Am. Chem. Soc. (1990) 112, 4960–4962; Erkkila, K. E. et al., Chem. Rev. (1999) 99, 2777–2795) The mechanism of this surprising effect has been elucidated to large degree. Luminescence quenching in aqueous solution seems to occur through water hydrogen bonding to dppz in the excited state, although solvent polarity may also play a role. (Olsen, E. J. C. et al., J. Am. Chem. Soc. (1997) 119, 11458–11467).
Another biomolecule of interest is nitric oxide (NO), a recognized ubiquitous biological second messenger molecule, that acts in a myriad of biological processes including neuronal development, regulation of blood pressure, apoptosis, neurotransmission, and immunological responses. (Kendrick, K. M. et al., Nature (1997) 388, 670–674; Huang, P. L. et al., Nature (1995) 377, 239–242; Ko, G. Y.; Kelly, P. T. J. Neurosci. (1999) 19, 6784–6794; Luth, H. J. et al., Brain Research (2000) 852, 45–55;Mize, R. R. et al., Nitric Oxide in Brain Development, Plasticity and Disease, Progress in Brain Research (Elesevier, 1998), vol. 118) (D. Nathan, J. Clin. Invest. (1997) 100, 2417–2423; J. Lancaster, Nitric Oxide: Principles and Actions (Academic Press, San Diego, Calif., 1996)). These diverse functions depend on the production of NO by nitric oxide synthase (NOS), a multidomain enzyme that catalyzes the overall transformation L-Arg+2O2+3/2(NADPH+H+)→L-citrulline+NO+2H2O+3/2 NADP+ where L-Arg is L-arginine and NADPH is nicotinamide adenine dinucleotide phosphate(Stuehr, D. J. Biochim. Biophys. Acta (1999) 1411, 217–230).
NO and NOS enzymes appear to play a role in many of the diseases that afflict humanity. This practical importance arises from the deep involvement of NOS in many of the channels of intercellular communication. During the 1990's considerable effort was expended in defining the characteristics of the various isoforms of NOS and their immediate effect on a wide array of cellular phenomena. Currently, the focus is shifting toward understanding how NOS functions within the context of the complex signaling pathways in and between cells. An example of this trend is the recent publication of a structural study of neuronal NOS that focused on the enzyme's interactions with PSD-95 and the NMDA receptor. (Hillier, B. J. et al., Science (1999) 284, 812–815)
The NOS monomer contains independently folded reductase and oygenase domains. The reductase domain binds NADPH and contains the cofactors FAD and FMN. The oxygenase domain contains a cysteine-ligated heme and a tetrahydrobiopterin (H4B) cofactor, and catalyses the oxidation of arginine to NO and citrulline. (Crane, B. R. et al., Science (1997) 278, 425–431; Crane, B. R. et al., Science (1998) 279, 2121–2126; Raman, C. S. et al., Cell (1998) 95, 939–950; Fischmann, T. O. et al., Nature Str. Biol. (1999) 6, 223–242) The oxygenase and reductase domains are joined by a calmodulin binding peptide that regulates the activity of the NOS isozymes. Interestingly, NOS functions as a dimer. Reduction occurs in trans—the reductase domain from one monomer reduces the oxidase domain of the complementary monomer. (Crane, B. R. et al., The EMBO Journal (1999) 18, 6271–6281)
The currently known mammalian NOS enzymes are organized into three classes: nNOS (neuronal), iNOS (immune), and eNOS (endothelial). These classifications reflect the origins of the NOS isoforms. (Bredt, D. S. et al., Nature (1991) 351, 714–718; Janssens, S. P. et al., J. Biol. Chem. (1992) 267, 22694; Lamas, S. et al., Proc. Natl. Acad. Sci. USA (1992) 89, 6348–6352; Lowenstein, C. J. et al., Proc. Natl. Acad. Sci. USA (1992) 89, 6711–6715; Xie, Q. W. et al., Science (1992) 256, 225–228) However, subsequent research has shown that the various forms of NOS occur in a wide variety of tissues, with a complex distribution.
Although nNOS is constitutively expressed, its level of expression is dynamically regulated. (Dawson, T. M. et al., Progress in Brain Research (1998) 118, 3–11) For example, nNOS activity is high in the developing olfactory and visual systems, but low in their mature counterparts. Abnormal nNOS activity has been implicated in a variety of diseases, including both Parkinson's and Alzheimer's disease. (Luth, H. J. et al., Brain Research (2000) 852, 45–55; Dawson, V. L.; Dawson, T. M. Progress in Brain Research (1998) 118, 215–229) The isozyme eNOS (endothelial NOS) is expressed in smooth muscles, including those lining blood vessels. (Huang, P. L. et al., Nature (1995) 377, 239–242) Local production of NO triggers the relaxation of the vascular tissue, leading to reduction in blood pressure. In addition to vasodilation, eNOS also modulates angiogenesis. (Dimmeler, S.; Zeiher, A. M Cell Death and Differentiation (1999) 6, 964–968) iNOS has both beneficial and destructive influences in the immune system. (D. Nathan, J. Clin. Invest. (1997) 100, 2417–2423) For instance, iNOS is thought to be essential in fighting Mycobacterium tuberculosis. (MacMicking, J. D. et al., Proc. Natl. Acad. Sci. USA (1997) 94, 5243–5248) However, iNOS is also involved in the often destructive inflammation response to infection or injury. (D. Nathan, J. Clin. Invest. (1997) 100, 2417-2423)
Despite the intense interest in nitric oxide synthases in the biological and medical community, aspects of the catalytic mechanism of these enzymes remain poorly understood. In particular, the function of the H4B cofactor has not been adequately explained.
Currently, two general methods are used for imaging NOS distribution. (Feelisch, M.; Stamler, J. S. Eds., Methods in Nitric Oxide Research (John Wiley and Sons, Inc., New York, 1996)) First, NADPH, arginine, NO, citrulline, nitrates, nitrates and other reactants and products of NOS can be detected chemically, usually through chemiluminescence or a stain (Kikuchi, K. et al., Analytical Chemistry (1993) 65, 1794–1799; Kojima, H. et al., Anal. Chem. (1998) 70, 2446–2453; Kishimoto, J. et al., Eur. J. Neuroscience (1993) 5, 1684–1694). Because these small molecules diffuse rapidly, this limits the spatial resolution of this technique. In addition, the staining techniques kill the sample. The chemiluminescence resulting from the reaction of NO with ozone can also be used to quantify NO production, but again gives limited spatial information. The second technique used is immunohistochemistry (Maines, M. D Ed., Nitric Oxide Synthase: Characterization and Functional Analysis (Academic Press, San Diego, Calif., 1996); Kobzik, L., Schmidt, H. H. H. W. in Methods in nitric oxide synthase M. Feelish, J. S. Stamler, Eds. (John Wiley and Sons, New York, 1996) pp. 229–236). Briefly, an antibody is raised against NOS. The antibody binds to NOS, and the antibody is then detected through staining or fluorescence. This gives better spatial resolution, but again the staining process destroys the sample.
The disadvantages of many of the prior known probes utilized to study biomolecules are their requirements for chemical or biological modification of the biomolecules for characterization. Furthermore, because Ru-substrates interact with their targets reversibly, they differ from current probes of heme proteins that rely on covalent modification and chemical analysis (Tschirret-Guth, R. A., et al. (1999) J. Am. Chem. Soc. 121, 4731–4737; Tschirret-Guth, R. A., et al. (1998) J. Am. Chem. Soc. 120, 7404–7410). The shortcomings of many presently available molecular probes for detecting biomolecules demonstrate the need for new agents that can detect and characterize biomolecules without the need for covalent or mutational modification of the protein of interest. The present invention satisfies this and other needs.